This proposal is centered on genetic and biochemical characterization of regulatory factors that control development of metabolically quiescent cysts in gram-negative bacteria. Numerous beneficial bacteria that fix nitrogen for plants, as well as harmful bacterial pathogens, are capable of synthesizing cysts. Cysts are metabolically dormant resting cells that are extremely resistant to desiccation as well as to growth inhibitors such as to antibiotics. This study analyzes cyst formation in the gram-negative model organism R. centenum for which numerous cyst developmental regulatory mutants have been isolated. One class of mutants dramatically overproduces cysts while a second class of mutants dramatically inhibits cyst formation. Molecular genetic analysis of mutant cell lines led to a surprising discovery that regulation of cyst formation involved production and excretion of cGMP. cGMP is a well known messenger in eukaryotes where it is involved in vision, muscle contraction, platelet aggregation, and development. However, its involvement in bacteria has until now been quite controversial. Our recent definitive studies have unequivocally demonstrated the involvement of cGMP in R. centenum cyst development and has implicated cGMP production in many other important bacterial species such as Azospirillum brasilense, Rhizobium leguminosarum, Mesorhizobium loti, Sinorhizobium medicae, Sinorhizobium meliloti, Sinorhizobium fredii, Delftia acidovorans (Comamonas acidovorans), Burkholderia mallei (Pseudomonas mallei)). Biochemical characterization of cGMP production, and its involvement in bacterial signal transduction, is a major goal of this proposal. We have also isolated numerous additional cyst developmental regulatory mutations that contain genetic disruptions in a variety of bacterial signal transduction components and in various transcription factors. Changes in genome wide gene expression will be undertaken with each of these mutants cell lines in order to develop detailed models of the regulatory circuits that are involved in controlling bacterial cyst formation. PUBLIC HEALTH RELEVANCE: This is a study of how bacteria synthesize dormant non-replicating resting cysts. Cyst cells care capable of remaining alive without replication for many decades and are resistant to drying and to growth inhibitors such as to antibiotics. Our study will shed light on how cells control the formation of cysts with a long term goal of controlling cyst formation.